Red cell differentiation is marked by dramatic changes in the morphology and biochemical composition of the erythroid membrane and underlying cytoskeleton. We have obtained evidence that at least one important component of the erythroid cytoskeleton, Protein 4. 1, is regulated by tissue and differentiation stage specific mRNA splicing in developing erythroblasts. Protein 4.1 in red cells is an 80 kd cytoskeletal protein needed to attach spectrin/actin filaments to the membrane by linking spectrin to the cytoplasmic domain of integral membrane proteins. Protein 4.1 consists of a complex family of isoforms in most tissues and species; these forms arise from a single genomic locus by alternative splicing of a common mRNA precursor. We have documented that expression of a specific exon that encodes the amino acids necessary for spectrin/actin binding is induced during red cell development. Expression of other sequences that seem to promote localization of isoforms to the nucleus is repressed. This process can be controlled in tissue culture by induction of erythroid maturation in mouse erythroleukemia cells (MELC). Our results suggest that alternative mRNA splicing is an important mechanism of gene regulation during erythroid differentiation. We propose to characterize the elements mediating the erythroid pattern of Protein 4.1 mRNA splicing. The genomic locus encoding the Protein 4.1 mRNA precursor will be cloned, and it's exon/intron structure defined. Minigenes will be constructed that span the introns and exons specifically selected or rejected for processing in erythroid and non-erythroid tissues. The constructs will be transfected into MELC and non-erythroid cells, and the comparative pattern of retained exons determined. Using site directed mutagenesis strategies, we shall then determine the minimum sequence elements required to generate these specific splicing patterns. During the latter stages of this project, in collaboration with the laboratory of Dr. Joan Steitz, we shall attempt to isolate the small nuclear RNAs and/or RNA binding proteins that provide the "trans" factors needed to regulate these splicing events. Our long-term goal is to characterize these factors, and study their regulation during red cell development.